(1) A survey of mammalian brain phosphoproteins as indigenous substrates for the calmodulin-dependent phosphatase, calcineurin, revealed three potential ones: G-substrate, DARPP-32, and Protein K-F. (2) Calcineurin was found to be activated by Ni(II) ions by as much as 40-fold in a time-dependent and irreversible manner. Direct, tight binding of N2 to calcineurin was demonstrated by the quenching of Trp-fluorescence of the enzyme by this metal ion. Both the activation and fluorescence quenching were not reversed by the addition of chelators such as EDTA and EGTA. Treatment of Ni-activated calcineurin with Chelex 100 and Sephadex G-50 columns revealed one tightly bound Ni ion per molecule of enzyme. The Ni effect may be physiologically important. (3) A search of effectors for calcineurin uncovered a group of inhibitors. Pyrophosphate was most effective with an inhibition constant of 1 MuM. Purine and pyrimidine dinucleotides were good inhibitors. The effect was not due to the chelation of indispensable metal ions. (4) Homogenous preparations of inhibitors of proteinase B lost their inhibitory ability upon incubation at pH5. The result confirms that this loss of inhibitory effect is due to conformational rearrangements of the inhibitors. (5) A calcium-inhibited phosphoprotein phosphatase was discovered in bovine brain. The inhibition was not mediated through calmodulin and was not relieved by other metal ions. The finds suggests that Ca regulates different phosphatases in a subtle and complex fashion.